Repeated LPS induces training and tolerance of microglial responses across brain regions

Background Neuroinflammation is involved in the pathogenesis of almost every central nervous system disorder. As the brain’s innate immune cells, microglia fine tune their activity to a dynamic brain environment. Previous studies have shown that repeated bouts of peripheral inflammation can trigger long-term changes in microglial gene expression and function, a form of innate immune memory. Methods and results In this study, we used multiple low-dose lipopolysaccharide (LPS) injections in adult mice to study the acute cytokine, transcriptomic, and microglia morphological changes that contribute to the formation of immune memory in the frontal cortex, hippocampus, and striatum, as well as the long-term effects of these changes on behavior. Training and tolerance of gene expression was shared across regions, and we identified 3 unique clusters of DEGs (2xLPS-sensitive, 4xLPS-sensitive, LPS-decreased) enriched for different biological functions. 2xLPS-sensitive DEG promoters were enriched for binding sites for IRF and NFkB family transcription factors, two key regulators of innate immune memory. We quantified shifts in microglia morphological populations and found that while the proportion of ramified and rod-like microglia mostly remained consistent within brain regions and sexes with LPS treatment, there was a shift from ameboid towards hypertrophic morphological states across immune memory states and a dynamic emergence and resolution of events of microglia aligning end-to-end with repeated LPS. Conclusions Together, findings support the dynamic regulation of microglia during the formation of immune memories in the brain and support future work to exploit this model in brain disease contexts. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-024-03198-1.


Supplementary Figure
: Temporal dynamics of cytokine expression after LPS treatment.RT-qPCR fold change comparisons for pro-inflammatory cytokines Il-1β and Tnf-α (*p<0.05,BH-corrected) from frontal cortex tissue isolated from female and male mice (A) 3 and 24 hours after LPS treatments or (B) at different timepoints after 1xLPS.Supplementary Figure S2: Differential gene expression analysis using lima-voom.(A) Reference for color scale for scaled gene expression in DEG heatmap in Figure 3. Counts per million (CPM) for each DEG was scaled within each brain region first, then scaled within genes across brain regions (rows) in the final heatmap (Fig. 3A).Breaks between each color of the heatmap scale were adjusted to fit the distribution of the scaled gene expression values such that 10% of the data was contained within each color break.

PBS vs 3xLPS_HC Volcano plot
Log Fold Change

A B C
Supplementary Figure S4: 2xLPS-sensitive and 4xLPS-sensitive cluster DEGs are enriched for microglia-relevant genes.(A) Significant enrichments against mouse microglial gene lists in MGEnrichment (36).Green circles denote a significant enrichment in the percent of DEGs for a given gene list compared to that of the background of all genes detected in the RNA-Seq experiment (grey circles).Only significant enrichments are shown (*p<0.01,BH-corrected).genes with higher expression in antibiotic treated mice compared to specific pathogen free mice at P60 (Thion et al., 2018) genes with higher expression in female antibiotic treated mice compared to specific pathogen free mice at P60 (Thion et al., 2018) genes with higher expression in female compared to male germ free mice at P60 (Thion et al., 2018) genes with higher expression in female compared to male specific pathogen free mice at P60 (Thion et al., 2018) genes with higher expression in female germ free mice compared to specific pathogen free mice at P60 (Thion et al., 2018) genes with higher expression in female specific pathogen free mice compared to antibiotic treated mice at P60 (Thion et al., 2018) genes with higher expression in female specific pathogen free mice compared to germ free mice at P60 (Thion et al., 2018) genes with higher expression in females compared to males at E18.5 (Thion et al., 2018) genes with higher expression in germ free mice compared to specific pathogen free mice at E18.5 (Thion et al., 2018) genes with higher expression in male antibiotic treated mice compared to specific pathogen free mice at P60 (Thion et al., 2018) genes with higher expression in male germ free mice compared to specific pathogen free mice at E18.5 (Thion et al., 2018) genes with higher expression in male specific pathogen free mice compared to germ free mice at E18.5 (Thion et al., 2018) genes with higher expression in specific pathogen free mice compared to germ free mice at P60 (Thion et al., 2018) homeostatic microglia cluster derived from yolk sac (E14.5)progenitors, P7 microglia and P60 microglia scRNAseq (Li et al., 2019) Injury response microglia collected from corpus callosum 7 days after focal demyelination of the subcortical white matter in mice is triggered by injection of lysolecithin (LPC (B) MDS plots colored by brain region and hemisphere along dimensions 1 and 2 and colored by treatment along dimensions 3, 4, and 5. (C) Distribution of log CPM for each sample before and after filtering.(D) Mean-variance trend of counts and final statistical model after limma-voom normalization.(E) Boxplots of Log2CPM values for each sample after TMM and limma-voom normalization.
DEGs within each brain region for each LPS versus PBS comparison.Volcano plots for Log Fold Change versus -log(corrected pvalue) for PBS versus each LPS treatment condition for (A) Frontal Cortex, (B) Hippocampus, and (C) Striatum.Genes with BH-corrected p-value < 0.05 and fold change>+/-2 for each comparison are colored red (increased) or blue (decreased) and labeled with the gene name.
Cluster 2a, b and c genes expressed predominantly at the youngest ages (E14.5 and P4/P5) and enriched for proliferation markers(Hammond et al, 2019)    Cluster 3 genes embryonic and early postnatal brain that expressed markers associated with metabolic activity, cell proliferation, cell growth, and cell motility(Hammond et al, 2019)    Cluster 4 Axon Tract−Associated Microglia (ATM) at PND4/5 resided only in the subcortical axon tracts of the corpus callosum in the forebrain, as well as in distinct clusters in the axon tracts of the cerebellum(Hammond et al, 2019)    Cluster 6 microglia reside in the embryonic brain parenchyma and bears considerable transcriptional overlap with brain border macrophages, Ms4a7+(Hammond et al, 2019)    Cluster OA2 Aging microglia that express inflammatory genes(Hammond et al, 2019)    Cluster OA3 Aging microglia that express interferon response genes(Hammond et al, 2019)    DEGs from IEG+ microglia compared to homeostatic microglia from 60 adult mice(Li et al., 2019)    Embryonic−like microglia identified at P7(Li et al., 2019)    Embryonic microglia cluster derived from yolk sac (E14.5)progenitors, P7 microglia and P60 microglia scRNAseq(Li et al., 2019)    Gene cluster with higher expression in adult microglia compared to other microglia developmental timepoints(Matcovitch−Natan, et al., 2016)    Gene cluster with higher expression in Yolk Sac progenitors compared to other microglia developmental timepoints(Matcovitch−Natan, et al., 2016) Density of foreground pixels in hull area Circularity Max/min radii from circle's center of mass Relative variation (CV) in radii from circle's center of mass Span ratio of hull (major/minor axis) Max/min radii from hull's center of mass Relative variation (CV) in radii from hull's center of mass # of slab voxels Foreground pixels Area Maximum radius from hull's center of mass Maximum span across hull Diameter of bounding circle Maximum radius from circle's center of mass Width of Decreased expression in control spinal microglial compared to injury activated spinal after treatment with Hdac3 inhibitor RGFP966(Wahane, et al., 2021)Decreased expression in isolated PND13 ARG1-YFP+CX3CR1+ microglia compared to ARG1-YFP-CX3CR1+ microglia from the area ventral to the corpus callosum and anterior to the lateral ventricles and excluding the olfactory bulb of P13 female and male(Stratoulias, et al, 2023)Differentially expressed genes with higher expression in Hdac1/2 KO MG compared to WT at Postnatal day 14; deletion embryonic(Datta, et al., 2018)Differentially expressed genes with higher expression in Hdac1/2 KO MG compared to WT at Postnatal day 45; deletion embryonic(Datta, et al., 2018)Differentially expressed genes with lower expression in Hdac1/2 KO MG compared to WT at embryonic day 16(Datta, et al., 2018)Differentially expressed genes with lower expression in Hdac1/2 KO MG compared to WT at Postnatal day 0; deletion embryonic(Datta, et al., 2018)Direct RNA sequencing of microglia from young and aged mice, top 100 genes showing ligand and microbial recognition to sense changes in the environment(Hickman, et al., 2013)genes with higher expression in cerebellum microglia compared to stratium microglia, TRAP isolation(Ayata, et al., 2018)genes with higher expression in Mef2cKO microglia comparted to control microglia(Deczkowska et al. 2018)genes with higher expression in microglia from mice in a germ free environment compared to standard housing(Erny, et al. 2015)genes with higher expression in stratium microglia compared to cerebellum microglia, TRAP isolation(Ayata, et al., 2018)higher expression in microglia from striatum with neuronal activationCNO (Badimon et al., 2020)Increased expression in Apoe4 microglia vs Apoe3 at 24 months of age(Lee et al., 2023)Increased expression in ApoeKO compared to WT microglia positive for apoptotic AlexaFluor488+ neurons(Krasemann et al., 2017)Increased expression in APPPS1:Trem2KO Tg compared to APPS1 Tg microglia male(Krasemann et al., 2017)Increased expression in injury activated spinal microglial compared to control spinal microglia(Wahane, et al., 2021)Increased expression in Ki67+ compared to Ki67− microglia at PND8 and after rrepopulation following PLX depletion(Belhocine et al., 2022)Increased expression in mice 48hr after brain injection of LPS (100ng) compared to vehicle(Krasemann et al., 2017)Increased expression in Non−phagocytosis + microglia compared to phagocytosis+ microglia(Krasemann et al., 2017)Increased expression in phagocytosis + microglia compared to non−phagocytosis+ microglia(Krasemann et al., 2017)Increased expression in SOD1 G93A mutant spinal microglia at endstage of disease (day 130) (ALS) compared to WT controls(Chiu et al., 2013)Increased expression in SOD1 G93A mutant spinal microglia at midstage of disease (day 100) (ALS) compared to WT controls(Chiu et al., 2013)Increased expression in Sod1:Trem2HET Tg compared to Sod1:Trem2KO microglia male(Krasemann et al., 2017)Increased expression in Sod1:Trem2KO Tg compared to Sod1:Trem2HET microglia male(Krasemann et al., 2017)Increased in expression in microglia fromAD (5XFAD), ALS (SOD1) and MS (EAE) due to response to neuronal death(Krasemann et al., 2017)Lipid droplet accumulating micrgolia (LDAM) gene signature ('LDhi' vs 'LDlo' DEGs; BODIPY microglia FACS)(Marschallinger et al.2019)PBS>LPSx4 6 month later whole brain DEGs(Wendeln et al., 2018)RNA sequencing acutely isolated microglia from females 2hours after systemic LPS or Saline injection(Hanamsagar et al., 2017)RNA sequencing acutely isolated microglia from males 2hours after systemic LPS or Saline injection(Hanamsagar et al., 2017)RNA sequencing male P80 isolated microglia, control, PolyIC MIA (GD 15), PolyIC MIA (GD 15) + minocycline (P70_P80 in drinking water)(Mattei, et al. 2017)Top 200 p adj genes defining microglia in Early response (clusters 2 vs 3)(Mathys et al., 2017)Top 200 p adj genes defining microglia in Early response (clusters 2 vs 3)(Zhou et al ., 2020)Weighted Gene Co−expression Network meta− analysis multiple microglial RNAseq datasets(Holtman et al., 2015)Weighted Gene Co−expression Network meta−analysis multiple microglial RNAseq datasets(Holtman et al., 2015)young adult MG, 4wks treatment choroid plexus AAV−Ctrl<AAV−INFb RNAseq(Deczkowska et al. 2018)

Supplementary Figure S6: Cxcl16 and S100a9 expression in microglia is dynamic in response to repeated LPS. (
).(Hammond et al, 2019)Postnatal immature microglia cluster derived from yolk sac (E14.5)progenitors,P7microglia and P60 microglia scRNAseq(Li et al., 2019)Postnatal immature microglia identified at P7(Li et al., 2019)Postnatal proliferative region−associated microglia (PAM) identified at P7 assoicated with white matter tracts(Li et al., 2019)Postnatal proliferative region−associated microglia (PAM) microglia cluster derived from yolk sac (E14.5)progenitors,P7microglia and P60 microglia scRNAseq(Li et al., 2019)Cell type expression of DEGs (BrainRNASeq.orgmouse data) A) Log2 fold change relative to PBS controls for each LPS treatment condition for Cxcl16 in RNAseq (top row) compared to RT-qPCR expression (bottom row) from the same mice.Patterns of expression were conserved across the two techniques for each brain region (Frontal Cortex, FC; Hippocampus, HC; and Striatum, STR).For RNAseq *p<0.05,BH-corrected for multiple comparison across all genes in the RNAseq.For RT-qPCR *p<0.05,corrected for multiple comparisons within the gene of interest.(B) Expression of Cxcl16 within different isolated brain cell types from Zhang et al., 2014 (https://web.stanford.edu/group/barres_lab/brain_rnaseq.html).(C) Expression of Cxcl16 by RT-qPCR in isolated cortical microglia and frontal cortex tissue following LPS treatments.*p<0.05,BH-corrected for multiple comparisons for 1-4xLPS treatment versus PBS.(D) Log2 fold change relative to PBS controls for each LPS treatment condition for S100a9 in RNAseq (top row) compared to RT-qPCR expression (bottom row) from the same mice.Patterns of expression were conserved across the two techniques for each brain region (Frontal Cortex, FC; Hippocampus, HC; and Striatum, STR).For RNAseq *p<0.05,BH-corrected for multiple comparison across all genes in the RNAseq.For RT-qPCR *p<0.05,corrected for multiple comparisons within the gene of interest.(E) Expression of S100a9 in isolated brain cell types from Zhang et al., 2014.(F) Expression of S100a9 by RT-qPCR in isolated cortical microglia and frontal cortex tissue following LPS treatments.*p<0.05,BH-corrected for multiple comparisons for 1-4xLPS treatment versus PBS.